nprot_2009_rnaseq

nprot_2009_rnaseq.markdown

###1

• process_name:preculture

• temperature: 32 celsius
• time : overnight
• comment : inoculating 30 ml of YES media
• input: yeast cell

• container: yeast agar plate

• input: yeast media
• output: precultured yeast

• container: 50ml flask

#2

• process_name:culture

• temperature: 32 celsius
• input: 1.1-1
• input: yeast media
• output:cultured yeast

#3

• process_name:separate culture

• input: 2.1-1
• output: separated cultured yeast
  • container: 50ml falcon tube
  • size:2

• process_name: centrifuge

• time: 2 min
• gravity: 1500 g
• temperature: 25 celsius
• comment : [critical step] Upregulation of stress genes can occur very rapidly in cells, so cells should be centrifuged and lysed as quickly as possible.
• input: 3.1-1
• output: separated cultured yeast with pellet

• comment: pellet formed

#4

• process_name:discard supernatant

• input: 3.2-1
• output: separated cultured yest with pellet without supernatant

• process_name:add

• input: 4.1-1

• target: True

• input: TES

• volume : 750 micro l

• output : cultured yest pellet with TES

• process: resuspend cell

• input : 4.2-1
• output : cultured yest with TES

#5

• process_name: add

• comment: Immediately
• input: 4.3-1
• target: True
• input: acidic phenol–chloroform

• volume: 750 micro l

• output: cultured yest with TES with acidic phenol–chloroform

• process_name: voltex

• time: 15s
• input: 5.1-1
• output: cultured yest with TES with acidic phenol–chloroform voltexed

#6

• process_name: preheat

• temperature: 65 celsius
• input: non heated heat block
• output : preheated heat block

#7

• process_name: incubate with voltex

• temperature: 65 celsius
• time: 1h
• comment: every 10 min 10s
• input: 5.2-1
• input: 6.1-1
• output: cultured yest with TES with acidic phenol–chloroform voltexed incubated

#8

• process: on ice

• time: 1 min
• input: 7.1-1
• input: ice
• output: cultured yest with TES with acidic phenol–chloroform voltexed incubated on ice

• process: voltex

• time: 20 s
• input: 8.1-1
• output: cultured yest with TES with acidic phenol–chloroform voltexed incubated valtexed

• process_name: centrifuge

• time: 15 min
• gravity: 14000 g
• temperature: 4 celsius
• input: 8.2-1
• output: cultured yest with TES with acidic phenol–chloroform centrifuged

#9

• process_name: centrifuge

• time: 10 s
• gravity: 14000 g
• temperature: RT
• input: 2 ml yellow phase-lock tube
• container: 2 ml yellow phase-lock tube
• output: 2 ml yellow phase-lock tube presipned

#10

• process_name: add

• input: 9.1-1
• target: True
• input: acidic phenol–chloroform

• volume: 700 micro l

• output: acidic phenol–chloroform + acidic phenol–chloroform

#11

• process_name: add

• input: 10.1-1
  • target: True
• input: 8.3-1
  • attribute: 700 micro l
• output: cultured yest + yellow tube

#12

• process_name: invert

• comment: thoroughly
• input: 11.1-1
• output: cultured yest + yellow tube inverted

• process_name: centrifuge

• time: 5 min
• gravity: 14000 g
• temperature: 4 celsius
• input: 12.1-1
• output: cultured yest + yellow tube inverted centrifuged

#13

• process_name: centrifuge

• time: 10 s
• gravity: 14000 g
• temperature: RT
• input: 2 ml yellow phase-lock tube
• container: 2 ml yellow phase-lock tube
• output: 2 ml yellow phase-lock tube presipned

#14

• process_name: add

• input: 13.1-1

• target:True

• input: chloroform:isoamyl alcohol (24:1)

• volume: 700 micro l

• output: 2 ml yellow phase-lock tube presipned + chloroform:isoamyl alcohol

#15

• process_name: add

• input: 14.1-1

• target: True

• input: 12.2-1

• volume: 700 micro l

• output : chloroform:isoamyl alcohol + yeast

#16

• process_name: invert

• comment: thoroughly
• input : 15.1-1
• output : chloroform:isoamyl alcohol + yeast mixed

• process_name: centrifuge

• time: 5 min
• gravity: 14000 g
• temperature: 4 celsius
• input : 16.1-1
• output : chloroform:isoamyl alcohol + yeast mixed centrifuged

#17

• process_name: add

• input: 2 ml Eppendorf tube
• container: 2 ml Eppendorf tube

• target: True

• input: ice cold 100% EtOH

• volume: 1.5 ml
• comment: ice cold

• output: 2 ml Eppendorf tube + EtOH

• process_name: add

• input: 17.1-1

• target: True

• input: 3 M NaAc pH 5.2

• volume: 50 micro l

• output: 2 ml Eppendorf tube + EtOH + NaAc

#18

• process_name: add

• input : 16.2-1

• volume: 500 micro l

• input: 17.2-1

• target: True

• output : chloroform:isoamyl alcohol + yeast + EtOH + NaAc

• process_name: voltex

• time: 10 s
• input : 18.1-1
• output: chloroform:isoamyl alcohol + yeast + etoh + naac voltexed

• process: precipitate

• temperature: -20 celsius
• time: overnight
• input: 18.2-1
• input: freezer
• output: chloroform:isoamyl alcohol + yeast + EtOH + NaAc + freezed

#19

• process_name: centrifuge

• time: 10 min
• gravity: 14000g
• temperature: RT
• input: 18.3-1
• output: chloroform:isoamyl alcohol + yeast + EtOH + NaAc + freezed centrifuged

• process_name: discard supernatant

• input: 19.1-1
• container: 2 ml Eppendorf tube
• output: yeast mix

• process_name: add attribute: 500 micro l

• input: 19.2-1
  • target : True
• input: 70% (vol/vol) EtOH

• temperature: 4 celsius
• comment: prepared with DEPC water

• output: yeast mix + 70% (vol/vol) EtOH

#20

• process_name: centrifuge

• time: 1 min
• gravity: 14000 g
• temperature: RT
• input: 19.3-1
• output: yeast mix + 70% (vol/vol) EtOH centrifuged

• process_name: discard supernatant

• input: 20.1-1
• output: yeast mix + 70% (vol/vol) EtOH centrifuged discarded

• process_name: centrifuge

• time: 5 s
• gravity: 14000 g
• temperature: RT
• input: 20.2-1
• output: yeast mix + 70% (vol/vol) EtOH centrifuged discarded centrifuged

• process_name: discard supernatant

• input: 20.3-1
• output: yeast mix + 70% (vol/vol) EtOH centrifuged discarded centrifuged discarded

#21

• process_name: air dry

• time: 5 min
• temperature: RT
• input: 20.4-1
• output: yeast dry pellet

#22

• process_name: add

• input: 21.1-1
  • target: True
• input: DEPC

• attribute: 100 micro l

• output: yeast + DEPC

• process_name: incubate

• time: 1 min
• temperature: 65 celsius
• input: 22.1-1
• input: heat block
• output: incubated yeast

• process_name: dissolve pellet

• comment: up and down more than 30 times
• comment: until no particles are left
• input: 20.2-1
• output: incubated yeast no pellet

• process_name: voltex

• time: 10 s
• comment: gently
• input: 20.3-1
• output: incubated yeast no pellet valotexed

#23

• process_name: measure RNA concentration

• reference: DEPC
• comment: Expect 1.6–4 mg of RNA (OD = 0.2–0.5)
• input: Nanodrop
• input: 22.4-1
• output: measured RNA amount
• output: incubated yeast no pellet valotexed

#24 ##つまった output: incubated yeast no pellet valotexed + good amount RNA container: 2 ml Eppendorf tube

#25

• process_name: incubate RNA attribute: RNase-free DNase

• time: 30 min
• temperature: 37 celsius
• comment: follow manufacturer’s instructions
• input: 24.1-1
• output: incubated yeast no pellet valotexed + good amount RNA incubated

• container: 2 ml Eppendorf tube

#26

• process_name: purify RNA

• comment: attribute: purify 100 μg of RNA
• input: Qiagen RNAeasy column
• input: 25.1-1
• output: yeast RNA

#27

• process_name: Assess the quality of RNA

• input: Agilent Bioanalyzer
• input: 26.1-1
• output: analyzed RNA data
• output: yeast RNA

#28

• process_name: poly(a) enrichment of RNA

• input: 27.1-1
• input: poly(A) + RNA selection kit
• output: poly(A)+ yeast RNA

#29

• process_name: cDNA preparation ( SuperScript double-stranded cDNA synthesis kit)

• input: 28.1-1
• input: SuperScript double-stranded cDNA synthesis kit
• ouput: yeast cDNA

#30

• process_name: DNA 1000 kit on the Agilent Bioanalyzer

• input: 29.1-1
• input: Agilent Bioanalyzer
• output: cDNA analyzed data
• output: yeast cDNA

#31

• process_name: add

• input: 30.1-1
• target: True
• input : TE

• volume: 50 micro l

• output: yeast cDNA + TE

• process_name: add

• input: 31.1-1

• target: True

• input: nebulization buffer (Illumina)
• output: yeast cDNA + TE + buffer

#32

• process_name: fragment

• condition: on_ice
• psi: 32–35 psi
• time: 6 min
• input: 31.2-1
• input: nebulizer
• output: fragmented yeast cDNA

#33

• process_name: Purify the fragmentation product (QIAquick kit)

• comment: eluting in 50 μl of water
• input: 32.1-1
• input: QIAquick kit
• output: purified yeast cDNA fragment

#34

• process_name: incubate cDNA fragment

• time: 30 min
• temperature: 16 celsius
• input: 33.1-1
• input: Klenow DNA polymerase

• U: 50U

• input: T4 DNA polymerase

• unit: 30 units

• input: dNTPs

• volume: 20 micro l
  • concentration: 10 mM

• output: blunted cDNA fragment

#35

• process_name: Purify cDNA fragment

• input: 34.1-1
• input: QIAquick PCR purification column
• input: Qiagen elution buffer
• volume: 32 mico l
• output: purified cDNA

#36

• process_name: add

• input: 35.1-1

• target: True

• input: exo-Klenow

• unit: 15 unit

• output: cDNA + exo-Klenow

• process_name: add

• input: 36.1-1
• target: True
• input: dATP

• volume: 10 micro l
  • concentration: 1 mM

• output: cDNA + exo-Klenow + dATP

• process_name: add

• input: 36.2-1

• target: True

• input: Klenow buffer

• volume: 5 micro l
  • concentration: 10 x

• output: cDNA + exo-Klenow + dATP + buffer

• process_name: incubate

• time: 30 min
• temperature: 37 celsius
• input: 36.3-1
• output: cDNA + adenine overhang

#37

• process_name: add

• comment: follow the current Illumina protocols
• input: 36.4-1
• target: True
• input: Add adaptors and T4 DNA ligase
• output: cDNA + adaptor

• process_name: incubate

• time: 15 min
• temperature: RT
• input: 37.1-1
• output: cDNA + adaptor + incubated #38

• process_name: Separate ligation products

• input: 37.2-1
• input: Tris-acetate-EDTA (TAE)-agarose gel

• concentration: 2% (wt/vol)

• output: cDNA on agarose gel

• process_name: excise region

• input: 38.1-1
• insterment: razor blade
• output: excised cDNA

#39

• process_name: gel purification

• comment: follow the manufacturer’s protocol
• input: 38.2-1
• input: Qiagen Gel purification kit
• input: water
• volume: 30 micor l
• output: purified cDNA

#40

• process_name: PCR

• comment: follow the manual
• input: 39.1-1
• target: True
• input: PCR machine
• input: water

• volume: 22 micro l

• input: 2X Phusion Taq master mix

• volume: 25 micor l

• input: 1.1 primer (Illumina)

• volume: 1 micro l

• input: 2.1 primer (Illumina)

• volume: 1 micro l

• output: PCR cDNA

#41

• process_name: purify the PCR product

• input: 40.1-1
• input : QIAquick PCR kit
• output: purified PCR product(cDNA)

• process_name: measure RNA concentration

• input: Nanodrop
• input: 41.1-1
• output: measured RNA amount
• output: purified PCR product(cDNA)

#42

• process_name: Dilute cDNA

• input: 41.2-1
• input: TE

• concentration: 10 nM

#43

• process_name: Illumina genome analyzer

• input: 42.1-1
• input: Illumina cluster station
• input: xml script
• output: analyzed Illumina data